Loss-of-function mutations in Lysyl-tRNA synthetase cause various leukoencephalopathy phenotypes

Objective: To expand the clinical spectrum of lysyl-tRNA synthetase (KARS) gene–related diseases, which so far includes Charcot-Marie-Tooth disease, congenital visual impairment and microcephaly, and nonsyndromic hearing impairment. Methods: Whole-exome sequencing was performed on index patients from 4 unrelated families with leukoencephalopathy. Candidate pathogenic variants and their cosegregation were confirmed by Sanger sequencing. Effects of mutations on KARS protein function were examined by aminoacylation assays and yeast complementation assays. Results: Common clinical features of the patients in this study included impaired cognitive ability, seizure, hypotonia, ataxia, and abnormal brain imaging, suggesting that the CNS involvement is the main clinical presentation. Six previously unreported and 1 known KARS mutations were identified and cosegregated in these families. Two patients are compound heterozygous for missense mutations, 1 patient is homozygous for a missense mutation, and 1 patient harbored an insertion mutation and a missense mutation. Functional and structural analyses revealed that these mutations impair aminoacylation activity of lysyl-tRNA synthetase, indicating that de- fective KARS function is responsible for the phenotypes in these individuals. Conclusions: Our results demonstrate that patients with loss-of-function KARS mutations can manifest CNS disorders, thus broadening the phenotypic spectrum associated with KARS-related disease

Phenotypic and molecular characterisation of CDK13-related congenital heart defects, dysmorphic facial features and intellectual developmental disorders

Background: De novo missense variants in CDK13 have been described as the cause of syndromic congenital heart defects in seven individuals ascertained from a large congenital cardiovascular malformations cohort. We aimed to further define the phenotypic and molecular spectrum of this newly described disorder. Methods: To minimise ascertainment bias, we recruited nine additional individuals with CDK13 pathogenic variants from clinical and research exome laboratory sequencing cohorts. Each individual underwent dysmorphology exam and comprehensive medical history review. Results: We demonstrate greater than expected phenotypic heterogeneity, including 33% (3/9) of individuals without structural heart disease on echocardiogram. There was a high penetrance for a unique constellation of facial dysmorphism and global developmental delay, as well as less frequently seen renal and sacral anomalies. Two individuals had novel CDK13 variants (p.Asn842Asp, p.Lys734Glu), while the remaining seven unrelated individuals had a recurrent, previously published p.Asn842Ser variant. Summary of all variants published to date demonstrates apparent restriction of pathogenic variants to the protein kinase domain with clustering in the ATP and magnesium binding sites. Conclusions: Here we provide detailed phenotypic and molecular characterisation of individuals with pathogenic variants in CDK13 and propose management guidelines based upon the estimated prevalence of anomalies identified. Keywords: CDK13, CHDFIDD, De novo variant, Neurodevelopmental disorders, Agenesis of the corpus callosum, Hypertelorism, Developmental delay, Cyclin-dependent kinase, Undiagnosed Diseases Networ

Phenotypic expansion in DDX3X - a common cause of intellectual disability in females

De novo variants in DDX3X account for 1-3% of unexplained intellectual disability (ID) cases and are amongst the most common causes of ID especially in females. Forty-seven patients (44 females, 3 males) have been described. We identified 31 additional individuals carrying 29 unique DDX3X variants, including 30 postnatal individuals with complex clinical presentations of developmental delay or ID, and one fetus with abnormal ultrasound findings. Rare or novel phenotypes observed include respiratory problems, congenital heart disease, skeletal muscle mitochondrial DNA depletion, and late-onset neurologic decline. Our findings expand the spectrum of DNA variants and phenotypes associated with DDX3X disorders

Human Dectin-1 Deficiency Impairs Macrophage-Mediated Defense Against Phaeohyphomycosis

Subcutaneous phaeohyphomycosis typically affects immunocompetent individuals following traumatic inoculation. Severe or disseminated infection can occur in CARD9 deficiency or after transplantation, but the mechanisms protecting against phaeohyphomycosis remain unclear. We evaluated a patient with progressive, refractory Corynespora cassiicola phaeohyphomycosis and found that he carried biallelic deleterious mutations in CLEC7A encoding the CARD9-coupled, β-glucan-binding receptor, Dectin-1. The patient\u27s PBMCs failed to produce TNF-α and IL-1β in response to β-glucan and/or C. cassiicola. To confirm the cellular and molecular requirements for immunity against C. cassiicola, we developed a mouse model of this infection. Mouse macrophages required Dectin-1 and CARD9 for IL-1β and TNF-α production, which enhanced fungal killing in an interdependent manner. Deficiency of either Dectin-1 or CARD9 was associated with more severe fungal disease, recapitulating the human observation. Because these data implicated impaired Dectin-1 responses in susceptibility to phaeohyphomycosis, we evaluated 17 additional unrelated patients with severe forms of the infection. We found that 12 out of 17 carried deleterious CLEC7A mutations associated with an altered Dectin-1 extracellular C-terminal domain and impaired Dectin-1-dependent cytokine production. Thus, we show that Dectin-1 and CARD9 promote protective TNF-α- and IL-1β-mediated macrophage defense against C. cassiicola. More broadly, we demonstrate that human Dectin-1 deficiency may contribute to susceptibility to severe phaeohyphomycosis by certain dematiaceous fungi

Increased insulin/insulin growth factor signaling advances the onset of metamorphosis in Drosophila.

Mechanisms by which attainment of specific body sizes trigger developmental transitions to adulthood (e.g. puberty or metamorphosis) are incompletely understood. In Drosophila, metamorphosis is triggered by ecdysone synthesis from the prothoracic gland (PG), whereas growth rate is increased by insulin/insulin growth factor signalling (IIS). Transgene-induced activation of PI3K, the major effector of IIS, within the PG advances the onset of metamorphosis via precocious ecdysone synthesis, raising the possibility that IIS triggers metamorphosis via PI3K activation in the PG. Here we show that blocking the protein kinase A (PKA) pathway in the insulin producing cells (IPCs) increases IIS. This increased IIS increases larval growth rate and also advances the onset of metamorphosis, which is accompanied by precocious ecdysone synthesis and increased transcription of at least one ecdysone biosynthetic gene. Our observations suggest that IIS is regulated by PKA pathway activity in the IPCs. In addition, taken together with previous findings, our observations are consistent with the possibility that, in Drosophila, attainment of a specific body size triggers metamorphosis via the IIS-mediated activation of PI3K and hence ecdysone synthesis in the PG

The regulation of ecdysone synthesis by insulin and PTTH.

<p>Brain lobes and the ring gland are indicated. Ecdysone synthesis is triggered when insulin released from the IPCs and PTTH released from the PG-LPs activate the PI3K and Ras pathways, respectively, in the PG. These two pathways, acting together, activate transcription of the PG-specific “Halloween genes”, which encode the ecdysone biosynthetic enzymes, ultimately triggering ecdysone synthesis. Arrows indicate activation pathways for which there is experimental evidence in Drosophila, hatched arrow indicates a speculative activation pathway.</p

Inhibition of the PKA pathway in the IPCs increases IIS.

<p>(A) Brain lobes and ring gland from <i>dilp2>YFP</i> wandering third instar larva, showing enrichment of anti-Creb immunoreactivity in the IPCs. Red: anti-Creb, green: YFP. The scale bar is 35 µm. (B) Mean larval weight (y-axis) of the indicated genotypes (x-axis) from 76 to 144 hours after egg laying (AEL). Error bars represent SEMs. At least 3 independent biological samples of each genotype, each containing at least 3 larvae, were measured. (C) Mean <i>Thor</i> transcript levels (y-axis) were determined from two biological samples collected from each genotype (x-axis) and measured in triplicate at the indicated time AEL. Data were obtained by quantitative RT-PCR with the relative 2^−ΔΔCt method using the Thor and RPII140 expression assay (Applied Biosystems). Error bars represent SEMs. (D) Western blots from protein extracts obtained from developmentally staged larvae (108 hours AEL) of the genotypes indicated below the gels, using the antibody indicated to the right of each gel. The top two panels and bottom two panels represent blots made from separate gels. (E) Female adult weight (y-axis) of the indicated genotypes (X-axis). At least six biological samples each containing at least five flies were measured. Means+/−SEMs are indicated. (F) Length (y-axis) of >10 female pupae of the indicated genotypes (X-axis). Means+/−SEMs are indicated. (G) Photographs of female pupae of the indicated genotypes.</p

Increasing IIS causes precocious metamorphosis.

<p>(A) Mean fraction of developmentally staged larvae pupariating (y-axis) by the indicated time after egg laying (AEL, x-axis). Error bars represent SEMs, genotypes are listed in the inset. At least six independent biological samples were measured for each data point. (B,C) Mean <i>E74B</i> and <i>dib</i> transcript levels (y-axis), respectively, were measured from two biological samples collected from developmentally staged larvae of the indicated genotype (x-axis) and measured in triplicate at the indicated time AEL. Error bars represent SEMs. Data were obtained by quantitative RT-PCR with the relative 2^−ΔΔCt method and normalized to <i>RpL13A</i>.</p